Rhodopsin activation exposes a key hydrophobic binding site for the transducin alpha-subunit C terminus. Export

@article{citeulike:1206481, abstract = {Conformational changes enable the photoreceptor rhodopsin to couple with and activate the G-protein transducin. Here we demonstrate a key interaction between these proteins occurs between the C terminus of the transducin alpha-subunit (G(Talpha)) and a hydrophobic cleft in the rhodopsin cytoplasmic face exposed during receptor activation. We mapped this interaction by labeling rhodopsin mutants with the fluorescent probe bimane and then assessed how binding of a peptide analogue of the G(Talpha) C terminus (containing a tryptophan quenching group) affected their fluorescence. From these and other assays, we conclude that the G(Talpha) C-terminal tail binds to the inner face of helix 6 in a retinal-linked manner. Further, we find that a "hydrophobic patch" comprising key residues in the exposed cleft is required for transducin binding/activation because it enhances the binding affinity for the G(Talpha) C-terminal tail, contributing up to 3 kcal/mol for this interaction. We speculate the hydrophobic interactions identified here may be important in other GPCR signaling systems, and our Trp/bimane fluorescence methodology may be generally useful for mapping sites of protein-protein interaction.}, address = {Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, USA.}, author = {Janz, J. M. and Farrens, D. L.}, citeulike-article-id = {1206481}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M402567200}, citeulike-linkout-1 = {http://www.jbc.org/cgi/content/abstract/279/28/29767}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/15070895}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=15070895}, day = {9}, doi = {10.1074/jbc.M402567200}, issn = {0021-9258}, journal = {J Biol Chem}, keywords = {paper1}, month = {July}, number = {28}, pages = {29767--29773}, posted-at = {2008-10-21 00:29:44}, priority = {2}, title = {Rhodopsin activation exposes a key hydrophobic binding site for the transducin alpha-subunit C terminus.}, url = {http://dx.doi.org/10.1074/jbc.M402567200}, volume = {279}, year = {2004} }

TY - JOUR ID - citeulike:1206481 L3 - citeulike-article-id:1206481 N2 - Conformational changes enable the photoreceptor rhodopsin to couple with and activate the G-protein transducin. Here we demonstrate a key interaction between these proteins occurs between the C terminus of the transducin alpha-subunit (G(Talpha)) and a hydrophobic cleft in the rhodopsin cytoplasmic face exposed during receptor activation. We mapped this interaction by labeling rhodopsin mutants with the fluorescent probe bimane and then assessed how binding of a peptide analogue of the G(Talpha) C terminus (containing a tryptophan quenching group) affected their fluorescence. From these and other assays, we conclude that the G(Talpha) C-terminal tail binds to the inner face of helix 6 in a retinal-linked manner. Further, we find that a "hydrophobic patch" comprising key residues in the exposed cleft is required for transducin binding/activation because it enhances the binding affinity for the G(Talpha) C-terminal tail, contributing up to 3 kcal/mol for this interaction. We speculate the hydrophobic interactions identified here may be important in other GPCR signaling systems, and our Trp/bimane fluorescence methodology may be generally useful for mapping sites of protein-protein interaction. IS - 28 JF - J Biol Chem SN - 0021-9258 AD - Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, USA. EP - 29773 TI - Rhodopsin activation exposes a key hydrophobic binding site for the transducin alpha-subunit C terminus. VL - 279 SP - 29767 KW - paper1 AU - Janz, JM AU - Farrens, DL PY - 2004/07/09/ UR - http://dx.doi.org/10.1074/jbc.M402567200 ER -